Method for detecting the presence of a single target nucleic acid in a sample

Abstract

A method comprising subjecting one or more sample portion(s) to a single amplification step, thereby amplifying a single molecule in the sample portion to a detectable level, and, in some embodiments, then determining whether the sample portion contains at least one molecule of the target nucleic acid. In some embodiments, the sample portion is in a porous sample structure, or in a sample chamber which comprises means for minimizing diffusion of the sample portion, or in a sample chamber which is inside a microcapillary device, or in a sample retaining means.

1. A method for amplifying a target nucleic acid present in a sample, the method comprising:
 combining a sample with reagents sufficient to perform a primer-based amplification reaction of at least one target nucleic acid when present; 
 introducing the combined sample and reagents along a passage of a microfluidic device; 
 introducing to the passage a fluid immiscible with the combined sample and reagents, wherein introducing the immiscible fluid segregates and entraps the combined sample and reagents into a plurality of reaction volumes ranging from 1 picoliter to 1 microliter, wherein said immiscible fluid is non-gaseous; and 
 collectively subjecting the plurality of reaction volumes to amplification conditions thereby amplifying the at least one target nucleic acid when present. 
 
     
     
       2. The method of  claim 1 , wherein the plurality of reaction volumes range from 1 picoliter to 100 nanoliters. 
     
     
       3. The method of  claim 1 , wherein the plurality of reaction volumes range from 1 picoliter to 10 nanoliters. 
     
     
       4. The method of  claim 1 , wherein the immiscible fluid is oil. 
     
     
       5. The method of  claim 1 , further comprising determining a quantity of the reaction volumes that yield amplified target nucleic acid from the collectively subjecting to the amplification conditions. 
     
     
       6. The method of  claim 1 , wherein the reagents comprise a detectable moiety sufficient to permit detection of the amplified target nucleic acid.